U.S. patent number 4,923,586 [Application Number 07/407,672] was granted by the patent office on 1990-05-08 for enzyme electrode unit.
This patent grant is currently assigned to Daikin Industries, Ltd.. Invention is credited to Hideo Katayama, Tatsuhiko Osaka, Yoshiaki Yoshida.
United States Patent |
4,923,586 |
Katayama , et al. |
May 8, 1990 |
Enzyme electrode unit
Abstract
An enzyme electrode unit having, on the surfaces of the base
electrodes thereof, an enzyme-immobilized membrane for oxidizing or
reducing a target substance to be measured, and a
diffusion-limiting membrane unit of a two-layer structure disposed
on the surface of the enzyme-immobilized membrane, only the
diffusion-limiting membrane having a lower target substance
diffusion limiting effect being replaceable, thus maintaining
substantially constant the general target substance diffusion
limiting effect of the diffusion-limiting membrane unit, even after
the replaceable diffusion-limiting membrane has been replaced.
Inventors: |
Katayama; Hideo (Kusatsu,
JP), Yoshida; Yoshiaki (Ikoma, JP), Osaka;
Tatsuhiko (Kurita, JP) |
Assignee: |
Daikin Industries, Ltd. (Osaka,
JP)
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Family
ID: |
26421444 |
Appl.
No.: |
07/407,672 |
Filed: |
September 14, 1989 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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176281 |
Mar 31, 1988 |
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Foreign Application Priority Data
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Mar 31, 1987 [JP] |
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62-80431 |
Mar 31, 1987 [JP] |
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62-80433 |
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Current U.S.
Class: |
204/403.05;
204/403.09; 204/403.11; 435/287.9; 435/817 |
Current CPC
Class: |
C12Q
1/001 (20130101); C12Q 1/006 (20130101); Y10S
435/817 (20130101) |
Current International
Class: |
C12Q
1/00 (20060101); C12M 001/34 () |
Field of
Search: |
;435/817,291
;204/1E,403 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Kaplan; G. L.
Attorney, Agent or Firm: Beveridge, DeGrandi &
Weilacher
Parent Case Text
This application is a continuation, of application Ser. No.
176,281, filed Mar. 31, 1988, now abandoned.
Claims
What is claimed is:
1. An enzyme electrode unit, comprising:
an enzyme-immobilized membrane;
a first diffusion-limiting membrane having a first target substance
diffusion limiting effect, said first membrane being secured to the
surface of said enzyme-immobilized membrane;
a second diffusion-limiting membrane having a target substance
diffusion limiting effect which is lower than said first target
substance diffusion limiting effect;
first holding means for holding in place said first
diffusion-limiting membrane and said enzyme-immobilized membrane;
and
second holding means for holding said second diffusion-limiting
membrane in a releasably disposed manner on a surface of said first
diffusion-limiting membrane
2. An enzyme electrode unit as set forth in claim 1, wherein each
of said diffusion-limiting membranes has a penetration ratio, and
the penetration ratio of the second diffusion-limiting membrane is
greater than the penetration ratio of the first diffusion-limiting
membrane.
3. An enzyme electrode unit as set forth in claim 1, wherein the
second diffusion-limiting membrane prevents substances having a
particle diameter greater than that of a target substance to be
measured, from penetrating therethrough.
4. An enzyme electrode unit as set forth in claim 1, wherein said
second holding means is a plastic cap.
5. An enzyme electrode unit as set forth in claim 1, wherein the
enzyme-immobilized membrane provokes an enzyme reaction of a target
substance in blood, and the second diffusion-limiting membrane
separates blood corpuscles.
6. An enzyme electrode unit as set forth in claim 1, wherein the
enzyme-immobilized membrane is a membrane in/on which glucose
oxidase is immobilized.
7. An enzyme electrode unit as set forth in claim 1, wherein the
target substance is glucose, and the glucose diffusion limiting
membrane effect of the first diffusion-limiting membrane is greater
than the glucose diffusion limiting effect of the second
diffusion-limiting membrane.
8. An enzyme electrode unit as set forth in claim 1, further
comprising a selective penetration membrane, and wherein the
enzyme-immobilized membrane is sandwiched in between said selective
penetration membrane and said first diffusion-limiting membrane,
and said selective penetration membrane, said enzyme-immobilized
membrane and said first diffusion limiting membrane are integrally
laminated and held as a unit.
9. An enzyme electrode unit as set forth in claim 8, wherein the
selective penetration membrane is stuck and secured to the
enzyme-immobilized membrane by a casting membrane method, and the
first diffusion-limiting membrane is bonded to the
enzyme-immobilized membrane.
10. An enzyme electrode unit as set forth in claim 8, wherein the
selective penetration membrane is a hydrogen peroxide selective
penetration membrane of hydrogen peroxide, the first
diffusion-limiting membrane is a diffusion-limiting membrane of
glucose, and the enzyme-immobilized membrane is a membrane in/on
which glucose oxidase is immobilized.
11. An enzyme electrode unit as set forth in claim 1, wherein said
second holding means is a resilient plate with an aperture formed
therein.
12. An enzyme electrode unit comprising:
an enzyme-immobilized membrane;
a first diffusion-limiting membrane having a first target substance
diffusion limiting effect, said first membrane being secured to the
surface of said enzyme-immobilized membrane;
a second diffusion-limiting membrane having a target substance
diffusion limiting effect which is lower than said first target
substance diffusion limiting effect;
first holding means or holding in place said first
diffusion-limiting membrane and said enzyme-immobilized membrane;
and
second holding means for holding said second diffusion-limiting
membrane disposed on a surface of said first diffusion membrane,
and said second holding means being releasably attached to said
first holding means.
13. An enzyme electrode unit comprising:
an enzyme-immobilized membrane;
a first diffusion-limiting membrane having a first target substance
diffusion limiting effect, said first membrane being secured to the
surface of said enzyme-immobilized membrane;
a second diffusion-limiting membrane having a target substance
diffusion limiting effect which is lower than said first target
substance diffusion limiting effect;
first holding means for holding in place said first
diffusion-limiting membrane and said enzyme-immobilized membrane;
and
second holding means for holding said second diffusion-limiting
membrane disposed on a surface of said first diffusion membrane,
and said second holding means including means for enabling the
release of said second diffusion-limiting membrane from a disposed
position on the surface of said first diffusion-limiting membrane
while maintaining the essential integrity of said first
diffusion-limiting membrane.
Description
BACKGROUND OF THE INVENTION
The present invention relates to an enzyme electrode unit, and more
particularly to an enzyme electrode unit in which
diffusion-limiting membranes limit the diffusion of a target
substance to be measured and the substance as limited in diffusion
is guided to an enzyme-immobilized membrane.
It is known that a physiologic active substance has a
characteristic capable of selectively detecting a very complicated
organic compound, protein or the like with high sensitivity. With
attention directed to this characteristic, researches and
developments have been made on measurement of such organic
compound, protein or the like with the use of an enzyme electrode
unit having base electrodes on which a physiologic active substance
is immobilized.
When measuring a target substance with the use of the enzyme
electrode unit above-mentioned, the target substance is oxidized or
reduced by the enzyme immobilized on the surfaces of the base
electrodes. By measuring the concentration of the oxygen, hydrogen
peroxide or the like which undergoes a change by such oxidization
or reduction, the concentration of the target substance can be
indirectly measured.
For example, when the concentration of glucose is to be measured,
glucose oxidase (hereinafter referred to as GOD) may be used as a
physiologic active substance. In this case, the following reaction
takes place: ##STR1## Accordingly, the concentration of glucose can
be determined by detecting the decrease in oxygen concentration or
the increase in hydrogen peroxide concentration.
More specifically, an enzyme-immobilized membrane having enzyme
immobilized on an acetylcellulose membrane is stuck to the surfaces
of hydrogen peroxide electrodes used as the base electrodes, and a
polycarbonate membrane covers the enzyme-immobilized membrane. To
enhance the sensitivity of measuring the concentration of glucose,
the total thickness of both membranes is set to 10 .mu.m.
In the example above-mentioned, the total membrane thickness is
extremely thin in order to enhance the concentration measuring
sensitivity. Consequently, the solution is guided to the
enzyme-immobilized membrane with the glucose concentration being
extremely high. Further, as apparent from the reaction formula
mentioned earlier, the glucose concentration measuring limit is
determined according to the amount of oxygen contained in a target
solution to be measured. As a result, the glucose concentration
measuring limit is very low. In this connection, if it is intended
to increase the glucose concentration measuring limit, it is
required to previously dilute the glucose solution at a
predetermined dilution ratio. This causes the dilution mechanism to
be complicated, and requires an expensive dilution apparatus.
To overcome such problems, it has been proposed, as disclosed in
the Japanese Laid-Open Patent Publication No. 59-22620, that a
diffusion-limiting membrane for limiting the diffusion of glucose
is disposed instead of the polycarbonate membrane to increase the
glucose concentration measuring limit without dilution of a glucose
solution.
The description hereinbefore which has discussed mainly the case of
measuring the concentration of glucose, may be also applied to the
case of measuring the concentration of other organic macromolecule,
protein or the like.
In the enzyme electrode unit above-mentioned, when a solution
containing a target substance to be measured also contains
interfering substances having a large particle diameter, the
diffusion-limiting membrane not only restricts the diffusion of the
target substance to be measured, but also prevents the interfering
substances from penetrating therethrough. This assures an accurate
measurement of a wide range of concentrations of a target substance
to be measured.
Upon completion of one measurement as above-mentioned, a relatively
great amount of interfering substances stick to the
diffusion-limiting membrane. This inevitably reduces that portion
of the diffusion-limiting membrane which achieves a predetermined
diffusion limitation for the target substance to be measured.
Therefore, the diffusion-limiting membrane as it is, cannot assure
an accurate measurement on and after the second operation.
Accordingly, it is a common practice that, after a predetermined
number of measurements has been made, preferably after every
measurement has been made, the diffusion-limiting membrane is
exchanged with a new one to achieve measurement without any
influence of the interfering substances. If there are neither
variations in the characteristics of the replaced
diffusion-limiting membranes themselves, nor variations in
diffusion-limiting membrane mounting condition, an accurate
measurement can be assured with the influence of the interfering
substances eliminated after the replacement of diffusion-limiting
membrane.
However, it is not assured at all that such variations are absent.
Generally, there exist not only considerable variations in the
characteristics of diffusion-limiting membranes themselves, but
also considerable variations in membrane mounting condition.
Accordingly, even though the influence of the interfering
substances can be eliminated, such variations may produce
considerable variations in measured results.
Further, the membrane mounted on the base electrodes is extremely
thin, requiring extreme care to be used when handling the
membrane.
It may be proposed that the diffusion-limiting membrane is mounted
on a cap or the like in consideration of its removal, and the
diffusion-limiting membrane is adapted to be automatically stuck to
the enzyme-immobilized membrane when such cap is secured to the
electrode unit body, threadedly or in other manner.
If a diffusion-limiting membrane is mounted in such manner, the
membrane has a limited portion for which physical adhesion is
assured. Accordingly, when a plurality of measurements are made
even without replacement of the diffusion-limiting membrane, the
measured data may considerably vary.
More specifically, when no measurement is still made, the
enzyme-immobilized membrane is held wet and the diffusion-limiting
membrane is also held wet. However, since no excessive electrode
conserving liquid is present, the adhesion of the
enzyme-immobilized membrane to the diffusion-limiting membrane is
assured fairly well throughout the surfaces. However, when
measurement starts by dropping a target solution to be measured on
the diffusion-limiting membrane or by dipping the enzyme electrode
unit in such solution, both membranes become excessively wet due to
the target solution to be measured. Accordingly, it is considered
that the adhesion of both membranes at other portions thereof than
those to which a physical pressing force is directly applied, may
be destroyed under the influence of surface tension or the like. It
is also considered that the adhesion of both membranes may be
destroyed under the influence of target solution dropping
conditions or conditions of dipping the enzyme electrode unit in
the target solution. The extent to which the adhesion of both
membranes is destroyed, varies in each measurement, resulting in
variations in measured data as above-mentioned.
Further, the diffusion-limiting membrane itself is very thin. This
requires extreme care to be used when replacing or handling the
membrane.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an enzyme
electrode unit capable of eliminating the influence of interfering
substances and considerably reducing the influence of variations
resulting from the replacement of a diffusion-limiting
membrane.
It is another object of the present invention to provide an enzyme
electrode unit capable of reducing variations in conditions of
mounting a diffusion-limiting membrane on an enzyme-immobilized
membrane.
It is a further object of the present invention to provide an
enzyme electrode unit in which the membranes to be mounted on the
base electrodes are easy to handle.
In order to achieve the objects above-mentioned, the enzyme
electrode unit in accordance with the present invention has base
electrodes for supplying an electric signal corresponding to the
amount of substance produced or consumed by a physiologic active
substance, and comprises;
an enzyme-immobilized membrane in/on which the physiologic active
substance is immobilized, secured to the surfaces of the base
electrodes;
a first diffusion-limiting membrane having a higher diffusion
limiting effect secured to the surface of the enzyme-immobilized
membrane; and
a second diffusion-limiting membrane having a lower diffusion
limiting effect removably disposed on the surface of the first
diffusion-limiting membrane.
Preferably, the enzyme-immobilized membrane has an obverse to which
the first diffusion-limiting membrane is stuck and secured, and a
reverse to which stuck and secured is a selective penetration
membrane through which the produced or consumed substance
selectively penetrates.
According to the present invention, a target substance to be
measured in a solution is guided to the enzyme-immobilized membrane
with the diffusion of the substance limited by the first and second
diffusion-limiting membranes, i.e., with the concentration of the
target substance lowered at a predetermined rate. Then, there may
be generated an electric signal corresponding to the concentration
of the target substance to be measured which has penetrated through
the diffusion-limiting membranes. Accordingly, the influence of
interfering substances may be eliminated and the concentration
measuring limit may be increased corresponding to the diffusion
limiting effect to the target substance to be measured.
When the second diffusion-limiting membrane is replaced, there may
exist variations in the characteristics of the second
diffusion-limiting membrane itself, as well as variations in
conditions of mounting the second diffusion-limiting membrane. In
spite of such variations, the general limiting effect of the first
and second diffusion-limiting membranes may be maintained
substantially constant. Further, the influence of interfering
substances may be effectively eliminated, thus assuring an accurate
measurement of the concentration of a target substance to be
measured.
More specifically, the general penetration ratio P of a unit of the
first and second diffusion-limiting membranes in its entirety, is
expressed by the following equation:
where
P1 is the penetration ratio of the first diffusion-limiting
membrane (this is a value which is obtained by dividing the
diffusion coefficient by the membrane thickness and which is
inversely proportional to the diffusion limiting effect), and
P2 is the penetration ratio of the second diffusion-limiting
membrane.
Since P2 is much greater than P1 (P2>>P1), the general
penetration ratio P undergoes no substantial change even though the
penetration ratio P2 varies more or less. Accordingly, even if the
second diffusion-limiting membrane is replaced in order to
eliminate the influence of obstructive substances, the general
diffusion limiting effect may be maintained substantially constant.
Thus, an accurate measurement of concentration may be assured.
When the enzyme-immobilized membrane has an obverse to which the
first diffusion-limiting membrane is stuck and secured, and a
reverse to which stuck and secured is the selective penetration
membrane through which the produced or consumed substance
above-mentioned selectively penetrates, it is possible to eliminate
the influence of the surface tension of a target solution to be
measured, the influence of conditions of such solution and the
like, enabling to obtain measured data with extremely small
variations.
All the membranes are held together as a unit, facilitating the
manipulation of the membranes.
Other objects, advantages and novel characteristics of the present
invention will be apparent from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a longitudinal section view of an enzyme electrode unit
in accordance with a first embodiment of the present invention;
FIG. 2 (A) shows measured data obtained with the use of the enzyme
electrode unit in FIG. 1;
FIG. 2 (B) shows measured data obtained with the use of a
comparative example of an enzyme electrode unit;
FIGS. 3 and 4 are electric circuit diagrams of electrode bias
devices applied to the enzyme electrode unit in accordance with the
present invention, respectively;
FIG. 5 is an exploded perspective view of an enzyme electrode unit
in accordance with a second embodiment of the present
invention;
FIG. 6 is a perspective view, with portions broken away,
illustrating how an GOD immobilized membrane is mounted on the
electrode unit of the present invention;
FIG. 7 is an exploded perspective view showing the relationship
between an undercap and a cap-like support member;
FIG. 8 is a perspective view illustrating a diffusion-limiting
membrane holding means on which a second diffusion-limiting
membrane is mounted;
FIG. 9 is a vertical section view of the center portion of the
means in FIG. 8; and
FIG. 10 is a schematic perspective view illustrating a mechanism
for positioning the diffusion-limiting membrane holding means in
FIG. 8.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 1 is a longitudinal section view of the enzyme electrode unit
in accordance with a first embodiment of the present invention.
The enzyme electrode unit includes: a rod-like member 1 made of an
insulating material; a center electrode made of Pt and an opposed
electrode 3 made of Ag, both electrodes 2 and 3 being disposed at
one surface of the rod-like member 1; a hydrogen peroxide selective
penetration membrane 4; a GOD immobilized membrane 5; a first
diffusion-limiting membrane 6; and a cap 7 for securing these
membranes 4, 5 and 6. The membranes 4, 5 and 6 are laminated on one
another as if covering that surface of the rod-like member 1 at
which both electrodes 2 and 3 are disposed. A second
diffusion-limiting membrane 8 is laminated on the first
diffusion-limiting membrane 6 with the use of a screw cap 9.
When measuring the concentration of glucose with use of the enzyme
electrode unit having the arrangement above-mentioned, it is
required to apply a predetermined bias voltage across the center
electrode 2 and the opposed electrode 3. In this connection, an
electrode bias device shown in FIG. 3 is used.
FIG. 3 is an electric circuit diagram of a first example of the
electrode bias device.
In the electrode bias device in FIG. 3, a current/voltage
converting resistance 52 is connected between an output terminal
51c and an inverting input terminal 51a of a current/voltage
converting operational amplifier 51, and a diode 53 is connected
between the inverting input terminal 51a and a non-inverting input
terminal 51b of the operational amplifier 51 such that the anode of
the diode 53 is connected to the inverting input terminal 51a. The
inverting input terminal 51a is connected to the center electrode 2
used as an anode-side electrode. The opposed electrode 3 used as a
cathode electrode is connected to a reverse bias voltage supply
terminal 55 through resistance 55a, and is also connected to
grounding 57 through a Zener diode 56. An output voltage from a
constant voltage supply source 58 is divided by resistances 58a,
58b, and the voltage thus divided is supplied to the non-inverting
input terminal 51b through a buffer amplifier 59. In the example
above-mentioned, since the hydrogen peroxide selective penetration
membrane 4 is used, the center electrode 2 is used as an anode-side
electrode while the opposed electrode 3 is used as a cathode-side
electrode. If an oxygen selective penetration membrane is used
instead of the hydrogen peroxide selective penetration membrane 4,
the center electrode 2 will be used as a cathode-side electrode
while the opposed electrode 3 will be used as an anode-side
electrode.
The following description will discuss the electrode bias operation
carried out by the electrode bias device having the arrangement
above-mentioned.
For measuring the concentration of glucose, a voltage of 0 V may be
supplied from the reverse bias voltage supply terminal 55, causing
the opposed electrode 3 to be grounded.
More specifically, the constant voltage supply source 58 always
generates a predetermined voltage, which is divided by the
resistances 58a, 58b (the divided voltage is set to 0.9 V for the
Pt-Ag electrodes). The divided voltage is supplied to the
non-inverting input terminal 51b of the current/voltage converting
operational amplifier 51 through the buffer amplifier 59.
Therefore, a reverse bias voltage is applied to the diode 53. Thus,
the divided voltage is applied to the center electrode 2 through
the inverting input terminal 51a by virtual grounding between the
inverting and non-inverting input terminals of the current/voltage
converting operational amplifier 51. That is, a forward bias
voltage is applied across the center electrode 2 and the opposed
electrode 3.
In such forward bias state, a current corresponding to the amount
of hydrogen peroxide produced by the reaction of glucose under the
presence of the GOD immobilized on the GOD immobilized membrane 5,
flows from the center electrode 2 to the opposed electrode 3. Since
the diode 53 is non-conductive, there can be taken out, from the
output terminal 51c of the current/voltage converting operational
amplifier 51, a voltage signal in which an offset voltage generated
by the forward bias voltage is being superposed on the voltage
signal proportional to the current above-mentioned.
By supplying the voltage signal thus taken out to a differentiation
circuit (not shown), the amount of voltage variation proportional
to the amount of output current variation can be obtained. Through
a predetermined operation of the amount of voltage variation thus
obtained, there is obtained the amount of output current variation
which corresponds to the glucose concentration.
It is known that, after the glucose concentration measuring
operation above-mentioned has been made, the oxidative reaction
forms an oxide layer on the electrode surfaces to decrease the
level of a signal taken out, assuring no accurate measurement of
glucose concentration. To prevent such decrease in signal level to
assure an accurate measurement of glucose concentration, a bias
voltage having a reversed polarity may be applied across the center
electrode 2 and the opposed electrode 3, thus removing the oxide
layer above-mentioned.
To carry out such oxide layer removing operation, a voltage (for
example, 5 V) higher than the divided voltage may be supplied from
the reverse bias voltage supply terminal 55.
More specifically, the divided voltage is supplied to the
non-inverting input terminal 51b of the current/voltage converting
operational amplifier 51 as done in the forward bias state.
Therefore, a reverse bias voltage is applied across the center
electrode 2 and the opposed electrode 3 through the Zener diode 56
(The reverse bias voltage is 1.2 V with the voltage across the
diode 53 terminals or the like taken into account).
In the reverse bias state, a current flows from the opposed
electrode 3 to the center electrode 2. When, with the increase in
current, the output from the current/voltage converting operational
amplifier 51 is saturated such that a voltage of the inverting
input terminal 51a becomes greater than a voltage of the
non-inverting input terminal 51b, a forward bias voltage is applied
to the diode 53, causing the same to be conductive. Accordingly, a
refresh current is bypassed by the diode 53. With a predetermined
reverse bias voltage applied across the center electrode 2 and the
opposed electrode 3, a sufficient amount of a current flows
therebetween, assuring a reliable refreshment operation.
Thereafter, a predetermined forward bias voltage is applied across
the center electrode 2 and the opposed electrode 3 as mentioned
earlier, whereby an accurate measurement of glucose concentration
can be made without influence of an oxide layer.
An example of the enzyme electrode unit according to the present
invention was formed. As the first diffusion-limiting membrane 6,
plain cellophane (type: #300) manufactured by Futamura Kagaku Kogyo
Co., Ltd. was used. As the second diffusion-limiting membrane 8,
there was used a polycarbonate membrane manufactured by Nuclepore
Corp. (bore diameter: 0.05 .mu.m, standard bore density:
6.times.10.sup.8 pores/cm.sup.2, flow amount of nitrogen: 0.8
liter/min/cm.sup.2 (10 psi), membrane thickness: 5 .mu.m).
With a bias voltage applied to the enzyme electrode unit having the
arrangement above-mentioned, measurements of glucose concentration
were made. FIG. 2 (A) shows the characteristics of the output
current variations/glucose concentrations obtained as the result of
such measurements. As a comparative example, measurements of
glucose concentration were made with an enzyme electrode unit using
a diffusion-limiting membrane made of cellophane only. FIG. 2 (B)
shows the characteristics of the output current variations/glucose
concentrations obtained as the result of such measurements. In the
comparative example in FIG. 2 (B), the output currents
corresponding to the same glucose concentrations are about twice
those in the Example in FIG. 2 (A). Accordingly, the enzyme
electrode in accordance with the present invention may have a
higher measuring limit of glucose concentration than that of the
comparative example.
With the use of the enzyme electrode having the arrangement
above-mentioned of the present invention, a solution having a
glucose concentration of 150 mg/dl was measured with the second
diffusion-limiting membrane 8 replaced for each measurement. The
following table shows the variations of output current in such
measurements. As a comparative example, with the use of an enzyme
electrode having one cellophane diffusion-limiting membrane, a
solution having a glucose concentration of 150 mg/dl was measured
with the cellophane replaced for every measurement. The output
current variations in the comparative example are also shown in the
following table.
TABLE
__________________________________________________________________________
Number of times 1 2 3 4 5 6 7 8 9 10
__________________________________________________________________________
Example 122 120 120 124 125 119 123 121 122 120 (nA/s) Comparative
162 207 191 186 216 223 170 218 220 221 Example (nA/s)
__________________________________________________________________________
As apparent from the Table, the average of the output current
variations in the Example is 122 nA/s, which is considerably small
as compared with the average 200 nA/s in the Comparative Example.
However, the variation throughout 10 measurements in the Example is
1.6%, which is considerably reduced as compared with the variation
10.9% in the Comparative Example. Accordingly, the Example
considerably reduces the variations in the general diffusion
limiting effect accompanied by the replacement of the second
diffusion-limiting membrane 8, thus achieving a stable measurement
of glucose concentration.
In particular, when measuring the glucose concentration in blood,
macromolecules such as albumen, blood corpuscles, enzyme or the
like may be stuck to the second diffusion-limiting membrane 8,
causing the same to get clogged. This requires replacement of the
second diffusion-limiting membrane 8 for each measurement. Even
under such conditions, the present invention assures a highly
stable measurement.
In the embodiment above-mentioned, the diffusion-limiting membrane
unit has a two-layer structure, of which only one
diffusion-limiting membrane presenting a smaller diffusion limiting
effect is replaceable. Accordingly, even though such
diffusion-limiting membrane is replaced, the general diffusion
limiting effect can be maintained substantially constant. This not
only assures a stable measurement of the concentrations of a target
substance, but also considerably reduces damages to the
enzyme-immobilized membrane resulting from the replacement of
diffusion-limiting membrane. Further, the replaceable
diffusion-limiting membrane may have a thickness which enables the
membrane to be easily wetted. This permits the replaceable
diffusion-limiting membrane to be preserved in a dry condition.
Moreover, the use of the electrode bias device mentioned earlier
assures an accurate measurement of a target substance with the
application of a forward bias voltage suffering no change by
external factors. Further, the diode is disposed to eliminate the
influence of saturation of the current/voltage converter with the
application of a reverse bias voltage for refreshment. A sufficient
amount of a refreshment current can let flow with the application
of a reverse bias voltage suffering no change by external
factors.
It was also found that, with the use of normal cellophane (Type:
#300) manufactured by Tokyo Cellophane Paper Co., Ltd. as the first
diffusion-limiting membrane 6, a stable measurement of glucose
concentrations was made.
FIG. 4 shows an electric circuit diagram of a second example of the
electrode bias device. The second example is the same as the first
example device in FIG. 3, except that a switching transistor 63 of
the npn type is used instead of the diode 53 in FIG. 3, and the
base terminal of the switching transistor 63 is connected to a
reverse bias voltage supply terminal 55 through resistance.
With such arrangement, the status of the switching transistor 63 is
forcibly controlled according to a voltage of the reverse bias
voltage supply terminal 55.
More specifically, when a voltage of 0 V is supplied from the
reverse bias voltage supply terminal 55, the switching transistor
63 becomes non-conductive. Accordingly, a predetermined forward
bias voltage can be applied across the center electrode 2 and the
opposed electrode 3, as in the first example in FIG. 3. In such
state, glucose concentrations can be measured.
On the contrary, when the reverse bias voltage supply terminal 55
supplies a voltage higher than a divided voltage, the switching
transistor 63 becomes conductive. A predetermined reverse bias
voltage is applied across the center electrode 2 and the opposed
electrode 3 as in the electrode bias device in FIG. 3. In such
state, the enzyme electrode unit can be refreshed.
In the electrode bias device having the arrangement in FIG. 4, a
pnp-type switching transistor or a field-effect transistor may be
used instead of the npn-type switching transistor 63.
FIG. 5 is an exploded perspective view of a second embodiment of
the enzyme electrode unit in accordance with the present
invention.
The second embodiment is the same as the first embodiment in FIG.
1, except that a hydrogen peroxide selective penetration membrane
4, a GOD immobilized membrane 5 and a first diffusion-limiting
membrane 6 are laminated on and securely stuck to one another.
More specifically, the hydrogen peroxide selective penetration
membrane 4 is stuck and secured to the GOD immobilized membrane 5
by a casting membrane method, and the diffusion-limiting membrane 6
is stuck and secured to the GOD immobilized membrane 5 by chitosan.
The membranes may be stuck and secured in other manner than that
above-mentioned, as far as the membranes can be sufficiently stuck
and secured to one another.
With the use of the enzyme electrode unit having the arrangement in
FIG. 5, a glucose concentration can be measured with higher
stability.
More specifically, a second diffusion-limiting membrane 8 has a
penetration ratio considerably higher than that of the first
diffusion-limiting membrane 6. Accordingly, even though both
diffusion-limiting membranes are not held together as a unit, the
membranes are not subject to substantial influence of the glucose
solution dropping conditions or the like. When the hydrogen
peroxide selective penetration membrane 4, the GOD immobilized
membrane 5 and the first diffusion-limiting membrane 6 are held
together as a unit, these membranes are considerably less
influenced by the glucose solution dropping conditions or the like
than the case where these membranes are not held together as a
unit.
As an Example, ten measurements of glucose concentration were made
with the hydrogen peroxide selective penetration membrane 4, the
GOD immobilized membrane 5 and the first diffusion-limiting
membrane 6 held together as a unit and without use of the second
diffusion-limiting membrane 8. The following table shows the output
current variations obtained in such measurements. As a Comparative
Example, ten measurements of glucose concentration were made with
the use of an enzyme electrode unit in which a diffusion-limiting
membrane 6 is secured to the GOD immobilized membrane 5 by a
cap.
TABLE
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Number of times 1 2 3 4 5 6 7 8 9 10
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Example 155 152 151 152 152 151 154 152 152 152 (nA/s) Comparative
157 168 169 171 169 169 174 175 181 179 Example (nA/s)
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As apparent from the Table, the average of measured data is 152
nA/s where the hydrogen peroxide selective penetration membrane 4,
the GOD immobilized membrane 5 and the first diffusion-limiting
membrane 6 were laminated and held together as a unit. This value
is smaller than the average 171 nA/s in the Comparative Example.
However, the variation in the measured data in the Example is
0.82%, which shows a considerable improvement in stability of
measured data as compared with the variation of 3.9% in the
Comparative Example.
As apparent from the second embodiment, the hydrogen peroxide
selective penetration membrane 4, the GOD immobilized membrane 5
and the first diffusion-limiting membrane 6 are laminated and held
together as a unit. Accordingly, a stable measurement can be
assured without influences of the surface tension of a glucose
solution, the glucose solution dropping conditions, the glucose
solution dipping conditions and the like.
FIG. 6 is a perspective view, with portions broken away, of an
example of an enzyme electrode unit in which a hydrogen peroxide
selective penetration membrane, a GOD immobilized membrane and a
first diffusion-limiting membrane are laminated and held together
as a unit. This enzyme electrode unit includes: a rod-like member 1
having a center electrode 2 and an opposed electrode 3; a hydrogen
peroxide selective penetration membrane 4; a GOD immobilized
membrane 5; a first diffusion-limiting membrane 6; a cap-like
support member 21; and an undercap 41 fitted in the cap-like
support member 21.
The rod-like member 1 has a hollow casing 17 made of an insulating
material such as synthetic resin. Lead lines 15, 16 are secured to
the inside of the casing 17 by filling the same with insulating
adhesives or the like. The center electrode 2 and the opposed
electrode 3 are embedded in and secured to one end surface of the
rod-like member 1. The opposed electrode 3 is provided in the outer
surface thereof with an external thread 19, with the use of which
the undercap 41 is mounted. The casing 17 is provided with an
external thread 18 in the outer surface at the electrode mounting
side thereof.
The center electrode 2 and the opposed electrode 3 are coaxial. The
rod-like member 1 has a convex curved surface (having the radius of
curvature of, for example, 10 mmR) at its end surface to which the
center electrode 2 and the opposed electrode 3 are secured.
As shown in FIG. 7, the undercap 41 has a casing 42 made of
polyacetal resins, which is provided at a predetermined position of
its external periphery with a projection 43 for controlling the
fitting amount of the cap-like support member 21. The undercap 41
has a GOD immobilized membrane holding surface 44 at the upper end
surface thereof.
As shown in FIG. 7, the cap-like support member 21 has a casing
body 22 made of polyacetal resins of which inner diameter is the
same as the outer diameter of the casing 42. The casing body 22 is
provided at the upper end thereof with an inwardly turned collar
integral therewith. The inwardly turned collar has a width greater
than that of the GOD immobilized membrane holding surface 44 of the
undercap 41. That portion of the inwardly turned collar which is
opposite to the GOD immobilized holding surface 44, serves as a GOD
immobilized holding surface 23. That portion of the inwardly turned
collar which is positioned at the inner part with respect to the
GOD immobilized holding surface 23, serves as a flange 24. The
collar is generally made thin such that the flange 24 can be
deformed along the convex curved surface of the rod-like member 1
when the cap-like support member 21 is fitted to the undercap
41.
FIG. 8 is a perspective view of diffusion-limiting membrane holding
means on which the second diffusion-limiting membrane 8 is mounted,
and FIG. 9 is a vertical section view of the center portion of the
means in FIG. 8.
This means has a relatively resilient thin plate 61 substantially
in the form of a rectangle which has resistance against a target
solution to be measured. The plate 61 has a square engagement
concave 62 at the center of one of the longer sides thereof. One
shorter side 63 is arcuate. The plate 61 has a circular opening 64
of which center is positioned at the center of a circle including
the arc. The second diffusion-limiting membrane 8 is attached to
the underside of the thin plate 61 with adhesives or by other
suitable means, such that the opening 64 is covered by this
membrane 8. That portion of the thin plate 61 which is close to the
other shorter side, serves as a holding portion 65.
As shown in FIG. 6, with the undercap 41 mounted on the rod-like
member 1, the integrally laminated membrane unit of the hydrogen
peroxide selective penetration membrane 4, the GOD immobilized
membrane 5 and the first diffusion-limiting membrane 6 is placed on
the convex curved surface of the rod-like member 1. Then, the
cap-like support member 21 is fitted to the undercap 41, causing
the integrally laminated membrane unit to be stuck to the convex
curved surface of the rod-like member 1.
More specifically, in the middle course of fitting the cap-like
support 21 to the undercap 41, the integrally laminated membrane
unit is held by and between the inner peripheral edge of the flange
24 and the convex surface of the rod-like member 1. At this time,
at least the center portion of the integrally laminated membrane
unit is stuck to the convex surface of the rod-like member 1.
However, it is not assured that the peripheral portion of the
membrane unit is entirely stuck to the peripheral portion of the
rod-like member 1. Generally, the membranes become more or less
creased. When fitting the cap-like support member 21 is continued,
the flange 24 is deformed along the convex surface of the rod-like
member 1. The point of the flange 24 which holds the integrally
laminated membrane unit together with the holding surface 44, is
gradually moved outward. The creases produced at the peripheral
portions of the membranes are thus removed. Finally, that portion
of the integrally laminated membrane unit which is positioned at
the inner side with respect to the flange 24, is completely stuck
to the convex surface of the rod-like member 1. Creases remains
only on those portions of the membranes which come in contact with
the GOD immobilized membrane holding surface 23 and the flange
24.
Then, the second diffusion-limiting membrane 8 mounted on the thin
plate 61 is stuck to the first diffusion-limiting membrane 6, as
shown in FIG. 10. This enables to maintain substantially constant
the general diffusion limiting effect of both diffusion-limiting
membranes 6 and 8, thus assuring an accurate measurement of glucose
concentration.
More specifically, the enzyme electrode unit is mounted at a
predetermined position on a base stand of a measuring apparatus
with the electrode surfaces projecting upward. The base stand has
engagement portions 71, 72 opposite to each other with the enzyme
electrode unit put therebetween. After said one shorter side 63 of
the thin plate 61 has been engaged with the engagement portion 71,
the holding portion 65 may be engaged with the engagement portion
72 by rotating the thin plate 61 with the holding portion 65 held
with the hand. The thin plate 61 is curved, causing the second
diffusion-limiting membrane 8 to be stuck to the first
diffusion-limiting membrane 6.
With the use of the mounting mechanism having the arrangement
above-mentioned, 100 measurements were made of a glucose solution
of which concentration is 150 mg/dl. The variation in measured data
was 3.2%, which demonstrates that the glucose concentration
measurements were made with high precision.
If the enzyme electrode unit has not been used for glucose
concentration measurement or has been preserved for a long period
of time to decrease the activity of the GOD immobilized membrane 5,
the second diffusion-limiting membrane 8 may be removed and the
integrally laminated membrane unit may be removed together with the
cap-like support member 21. Then, a new integrally laminated
membrane unit may be mounted under tension with the the use of the
cap-like support member 21, and the diffusion-limiting membrane 8
may be mounted again. Thereafter, a good glucose concentration
measurement may be assured.
When the mounting mechanism above-mentioned is used, it is possible
to entirely stick that portion of the integrally laminated membrane
unit which is exposed outside, to the convex surface of the
rod-like member throughout the unit surface by merely mounting the
cap-like support member 21. This results in improvements in
accuracy of a glucose concentration measurement.
The application of the mounting mechanism above-mentioned is not
limited to that for mounting an integrally laminated membrane unit,
but this mechanism may be also used for mounting membranes which
are not integrally laminated and held as a unit.
Instead of the hydrogen peroxide selective penetration membrane 4,
an oxygen selective penetration membrane may be used. In such case,
a glucose concentration may be measured based on the amount of
oxygen consumed as a result of an enzyme reaction.
The embodiments have been discussed in connection with measurement
of glucose concentration. However, instead of the GOD immobilized
membrane, there may be used an enzyme-immobilized membrane on which
another physiologic active substance than glucose oxidase is
immobilized. In such case, concentration measurement of other
organic macromolecule, protein or the like may be made.
With the use of the diffusion-limiting membrane holding means in
which the second diffusion-limiting membrane 8 is mounted on the
thin plate 61, the second diffusion-limiting membrane 8 may be
mounted on the enzyme electrode unit in FIG. 1.
* * * * *